Botanical Studies (2007) 48: 63-70. *Corresponding author: E-mail: [email protected]; Phone: +886-2-2736-1661 ext. 6160; Fax: +886-2-2378-0134. Introduction Yam (Dioscorea species) is a member of the mono- cotyledonous family Dioscoreaceae and is a staple food in West Africa, Southeast Asia, and the Caribbean (Akoruda, 1984). The fresh tuber slices are widely used as functional foods in Taiwan, and the dried slices are used as traditional Chinese medicines (Liu et al., 1995). Yam tuber contains mucilages, mannan-protein macromolecules (Misaki et al., 1972; Tsai and Tsai, 1984). Recently, yam tuber mucilage was reported to exhibit antioxidant (Hou et al., 2002; Lin et al., 2005), angiotensin converting enzyme inhibitory activities (Lee et al., 2003) and hypoglycemic activities (Hikino et al., 1986; Bailey and Day, 1989). Furthermore, Chinese yam (D. alata cv. Tainong No. 2) feeding re- sulted in antioxidant effects in hyperhomocysteinemia rats (Chang et al., 2004). Many isolated polysaccharides are reported to have immunomodulatory activities (Brown and Gordon, 2003; Feizi, 2000), and medicinal mushrooms (Wasser, 2002) have been intensively investigated for their beneficial ef- fects as immunomodulatory and antitumor agents. Len- tinan (Len), the (1→3)-β-glucan isolated from Lentinus edodes, has been demonstrated to have an anti-tumor activity against Sarcoma 180 in vivo and in vitro (Zhang et al., 2005). Reishi (Ganoderma lucidum) polysaccha- rides were reported as immune potentiators (Chang and Lu, 2004; Zhu and Lin, 2005; Hsu, et al., 2004). The cold- water extracts of dietary mushrooms, including Hypsizigus mamoreus, Agrocybe aegerita, and Flammulina velutipes, were showed to have antiproliferative activity against human leukemic U937 cells (Ou et al., 2005). The im- munomodulatory activity by an isolated α-glucan-protein complex from mycelium of Tricholoma matsutake has also been documented (Hoshi et al., 2005). Several food-grade microalgae, including Spirulina platensis, Aphanizomenon flos-aquae, and Chlorella pyrenoidosa, are also known to contain polysaccharides, potent immunostimulators of hu- man monocytes and macrophages (Pugh et al., 2001). In this study, orally administered mucilages from three dif- ferent Taiwanese yam cultivars, including Dioscorea alata L. cv. Tainong 1 (TN1), Dioscorea alata L. cv. Tainong 2 Immunostimulatory activities of yam tuber mucilages Huey-Fang SHANG 1 , Huey-Chuan CHENG 2 , Hong-Jen LIANG 3 , Hao-Yu LIU 4 , Sin-Yie LIU 5 , and Wen-Chi HOU 4, * 1 Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan 2 Mackay Memorial Hospital, Taipei 104 and Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan 3 Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, Taiwan 4 Graduate Institute of Pharmacognosy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 110, Taiwan 5 Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wu-Feng, Taichung, Taiwan (Received May 9, 2006; Accepted August 15, 2006) ABSTRACT. The purified mucilages from three Taiwanese yam cultivars, including Dioscorea alata L. cv. Tainong 1 (TN1), D. alata L. cv. Tainong 2 (TN2), and D. alata L. var. purpurea (Roxb.) cv. Ming-Jen (MJ), and the commercial lentinan (Len) were used to evaluate the immunostimulatory effects on the murine innate and adaptive immunity. BALB/c mice were grouped and administrated orally with 0.5 ml of TN1, TN2, MJ daily for 5 weeks. The positive and negative controls were fed with lentinan and distilled water, respectively. Blood samples were drawn from the retroorbital sinus on day 7 and 21, and the lymphocyte subpopulation, phagocytosis of granulocyte and monocyte were analyzed by flow cytometry. The mice were sacrificed on day 36, and the splenocytes were harvested for determinations of natural killer (NK) cell cytotoxicity activ- ity. The stimulation index on the phagocytosis of peritoneal macrophages and the RAW264.7 cell line by yam mucilage were also determined in vitro. The results showed that all three mucilages, especially MJ yam, could elevate the number of T helper cells in the peripheral blood and enhance the phagocytic activity of granulo- cyte, monocytes and macrophages both ex vivo and in vitro tests. Increased splenic cytotoxic activity follow- ing the administration of mucilages from MJ yam was observed. Furthermore, the production of specific anti- ovalbumin (OVA) antibody and OVA-stimulated splenic cell proliferation were also enhanced by all mucilage groups. It is suggested that the tuber mucilage may function as an immunomodulatory substance. Keywords: Dioscorea; Immunostimulatory; Lentinan; mucilage; Nk cell; Phagocytic activity; Yam. BIOCHEMISTRY
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Immunostimulatory activities of yam tuber mucilages
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1 Department of Microbiology and Immunology, Taipei Medical University, Taipei, Taiwan2 Mackay Memorial Hospital, Taipei 104 and Mackay Medicine, Nursing and Management College, Taipei 112, Taiwan3 Department of Food Science, Yaunpei University of Science and Technology, Hsinchu 300, Taiwan4 Graduate Institute of Pharmacognosy, Taipei Medical University, No. 250, Wu-Hsing Street, Taipei 110, Taiwan5 Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, Wu-Feng, Taichung, Taiwan
(Received�May�9,�2006;�Accepted�August�15,�2006)
ABSTRACT. The purified mucilages from three Taiwanese yam cultivars, including Dioscorea alata�L.�cv.�Tainong�1�(TN1),�D. alata�L.�cv.�Tainong�2�(TN2),�and�D. alata�L.�var.�purpurea�(Roxb.)�cv.�Ming-Jen�(MJ),�and�the�commercial�lentinan�(Len)�were�used�to�evaluate�the�immunostimulatory�effects�on�the�murine�innate�and�adaptive� immunity.�BALB/c�mice�were�grouped�and�administrated�orally�with�0.5�ml�of�TN1,�TN2,�MJ�daily�for�5�weeks.�The�positive�and�negative�controls�were�fed�with�lentinan�and�distilled�water,�respectively.��Blood� samples� were�drawn� from� the� retroorbital� sinus�on�day�7� and� 21,� and� the� lymphocyte� subpopulation,�phagocytosis� of� granulocyte� and� monocyte� were� analyzed� by� flow� cytometry.�The�mice�were� sacrificed� on�day� 36,� and� the� splenocytes� were� harvested� for� determinations� of� natural� killer� (NK)� cell� cytotoxicity� activ-ity.�The�stimulation�index�on�the�phagocytosis�of�peritoneal�macrophages�and�the�RAW264.7�cell�line�by�yam�mucilage�were�also�determined�in vitro.�The�results�showed�that�all�three�mucilages,�especially�MJ�yam,�could�elevate� the�number�of�T�helper�cells� in� the�peripheral�blood�and�enhance� the�phagocytic�activity�of�granulo-cyte,�monocytes�and�macrophages�both ex vivo�and� in vitro� tests.� Increased�splenic�cytotoxic�activity�follow-ing the administration of mucilages from MJ yam was observed. Furthermore, the production of specific anti-ovalbumin�(OVA)�antibody�and�OVA-stimulated�splenic�cell�proliferation�were�also�enhanced�by�all�mucilage�groups.�It�is�suggested�that�the�tuber�mucilage�may�function�as�an�immunomodulatory�substance.
ta�L.�cv.�Tainong�1�(TN1),�D. alata�L.�cv.�Tainong�2�(TN2),�and�D. alata L.�var.�purpurea (Roxb.)�cv.�Ming-Jen�(MJ),�were�kindly�provided�by�Dr.�Liu,�Sin-Yie�(Taiwan�Agricul-tural�Research�Institute,�Wu-Feng,�Taichung,�Taiwan).�The�TN1 tuber is a round or elliptic shape with white fleshes in�the�brown�peel.�The�TN2�tuber�is�a�cylindraceous�shape�with white fleshes in the brown peel. The MJ tuber is a cylindraceous shape with purple fleshes in the purplish-red peel.
Extraction and purification of yam tuber mucilage
After�washing�and�peeling,�the�yam�tubers�were�cut�into�strips for mucilage extraction and purification as described elsewhere (Hou et al., 2002; Lee et al., 2003). Briefly, yam tuber�was�homogenized� with� four� volumes� (W/V)� of� 50�mM�Tris-HCl� buffer� (pH� 8.3)� containing� 1%� vitamin� C.�After�centrifugation�at�14,000�×g�for�30�min,�the�superna-tants were mixed with isopropanol to a final concentration of�70%,�and�stirred�quickly�at�4ºC�overnight.�The�precipi-tates were filtrated and dehydrated with 100% isopropanol, then,�rinsed�with�acetone.�After�drying�at�40ºC�in�an�oven,�the� crude� mucilage� (CM)� was�ground� and� collected� for�further purification by both SDS and heating procedures. One�gram�of�CM�powder�was�dissolved�in�200�ml�distilled�water�and�kept�in�a�50ºC�water�bath.�Forty�mililiters�of�5%�SDS� solution� (dissolved� in� 45%� ethanol)� were� added� to�the�CM�solution.�The�mixture�was� stirred�gently�at�50ºC�for� 30�min,� then,� at� room� temperature� for� another� 2� h.�The�mucilage� solution� was� then� placed� in� an� ice� bath� to�quickly�reduce�the�temperature�in�order� to�precipitate� the�SDS-protein� complex.�After� centrifugation� as� above,� the�supernatants�were�precipitated�with�isopropanol�and�dried�in� a� 40ºC�oven� as� described� earlier.�The�mucilage�was�again�ground,�dissolved,�and�then�heated�in�boiling�water�for� 20� min.�After� centrifugation,� the� supernatants� were�mixed with isopropanol to a final concentration of 70%. The partially purified mucilage was filtrated, dehydrated, rinsed� with� acetone,� dried,� and� then� collected� for� further�use.�
from�National�Laboratory�Animal�Center�(Taipei,�Taiwan)�and divided randomly into five groups (n=8). Each group was� housed� individually� in�wire-bottomed� stainless� steel�cages� in� a� temperature-� and� humidity-controlled� room�(at� 22ºC)�with� a� 12-h� light/dark� cycle� and� free� access� to�AIN-76� feeds� and�water.�All� animal� experimental� proce-dures�followed�the�published�guidelines�(COA,�2004).�For�assessment�of�innate�immunity,�each�0.5�ml�of�TN1,�TN2,�MJ�(10�mg/m)�and�commercial�Len�(0.05�mg/ml)�were�ad-ministrated�orally�once�a�day�for�5�weeks.�Distilled�water�was�used�for�the�control�group.�Blood�samples�were�drawn�from�the�retroorbital�sinus�on�days�7�and�21,�and�the�lym-phocyte�subpopulation�and�both�phagocytosis�of�granulo-cyte, and monocyte were analyzed by flow cytometry as described�below.
or�MJ�yam�mucilages-treated�animals�is�increased�as�com-pared with the control group in the first week (Figure 1A) and�MJ�yam�mucilages-treated�ones�in�the�third�week�(Fig-ure�1B)�(P<0.01).�This�cell�increment�was�due�to�elevated�number�of�T�helper�cells�and�T�cytotoxic�cells�(Figures�1C�and�1D)�(P<0.05).�The�increased�T�helper�cells�were�also�found�after�glutamine�supplements�in�rats�with�gut-derived�sepsis� (Yeh� et� al.,� 2004).�Sinclair� (1998)� found� that� the�
66 Botanical Studies, Vol. 48, 2007
the third, but not the first week as compared with the con-trol� (P<0.01).� In� the� third� week� (Figure� 2B),� the� phago-cytic� activities� of� granulocyte� and�monocyte� populations�significantly increased in the Len (P<0.01),�TN1�(P<0.05),�TN2�(P<0.05),�and�MJ�(P<0.01)�groups.�Our�present�data�reveal� that� the�oral� administration�of� native�Taiwanese�yam�mucilages�can�elevate�the�phagocytic�cell�populations�of�BALB/c�mice�ex vivo.� In�mammals,�phagocytosis� is�a�very� important� defense� against� pathogen� invasions� and�apoptotic�cell� scavenging,�which� is�performed�by�phago-cytes� like�macrophages,� dendritic� cells,� and�granulocytes�(Stuart�et�al.,�2005).�Monocytes�and�other� leukocytes�are�recruited to the inflammatory site and differentiate in ad-vance to inflammatory macrophages (Van den Berg et al., 2001).
Phagocytosis of granulocyte and monocyte isolated from blood of BALB/c mice
In the first week (Figure 2A), the phagocytic activity of the�granulocyte�population�showed�no�apparent�differenc-es� from� the� control� group.�However,� phagocytic� activity�by�monocyte� population� significantly� increased� in�TN1,�TN2,�and�the�MJ�groups�as�compared�with�that�in�the�con-trol� (P<0.05�for�TN1�And�TN2�and�P<0.01�for�MJ).�The�phagocytic�activity�in�Len-treated�animals�was�elevated�in�
Figure 1.�The�effects�of�yam�mucilages�on�the�lymphocyte�subpopulations.�The�blood�from�the�retroorbital�sinus�on�day�7�and�day�21�was analyzed for both T and B cells (A and B) and T cell subsets (C and D) using flow cytometry. Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05�(*)�or�P<0.01�(**).�Bar�rep-resents�standard�deviation.
Figure 2.�The�effects�of�yam�mucilages�on�the�phagocytosis�of�granulocyte�and�monocyte�on�day�7�(A)�and�day�21�(B).�Means�of�trip-licates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05�(*)�or�P<0.01�(**).�Bar�represents�standard�deviation.
SHANG et al. — Immunostimulatory activities of yam tuber mucilages 67
Phagocytosis of peritoneal macrophage and the RAW264.7 cell line
Figure 3.�The�effects�of�yam�mucilages�on� the�phagocytic�activity�of�peritoneal�macrophage� (A)�and� the�RAW264.7�cell� line� (B).�Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05�(*)�or�P<0.01�(**).��Bar�represents�standard�deviation.
Figure 5. Effects of yam mucilages on the production of the serum specific anti-ovalbumin antibody of IgM (A) or IgG (B). Means of triplicates were measured. A difference between the control and each treatment was considered statistically significant when P<0.05�(*)�or�P<0.01�(**).�Bar�represents�standard�deviation.
SHANG et al. — Immunostimulatory activities of yam tuber mucilages 69
Gaforio,� J.J.,�M.J.�Serrano,�E.�Ortega,�L.�Algarra,� and�G.A.�de�Cienfuegos. 2002. Use of SYTOX green dye in the flow cy-tometric�analysis�of�bacterial�phagocytosis.�Cytometry�48: 93-96.�
Mlčková, P., D. Čechová, P. Chalupná, O. Novotná, and L. Prokešová.�2001.�Ehanced�systemic�and�mucosal�antibody�responses� to� a�model� protein� antigen� after� intranasal� and�intratracheal� immunization�using�Bacillus firmus�as�an�ad-juvant.�Immunol.�Lett.�77: 39-45.�
Tsai,�S.S.�and�F.J.�Tai.�1984.�Studies�on�the�mucilage�from�tuber�of�yam�(Dioscorea alata Linn.) I. Isolation and purification of�the�mucilage.�J.�Chin.�Agri.�Chem.�Soc.�22: 88-94.